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Development of a high throughput cell-based assay to screen for inhibitors of HIV-1 Vpr oligomerization

Zych, Courtney (2011) Development of a high throughput cell-based assay to screen for inhibitors of HIV-1 Vpr oligomerization. Master's Thesis, University of Pittsburgh. (Unpublished)

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Abstract

Highly active anti-retroviral treatment (HAART) targets less than a third of the proteinsproduced during HIV-1 infection. Testing the effectiveness of an anti-retroviral drug requiresassays specific for the individual target that take into account its mechanism of action. MostHIV-1 proteins need to undergo dimerization in order to become functional in the viral life cycle. Historically, it has been difficult to visualize and quantify changes in a protein-protein interaction, which has left this characteristic of proteins unexplored as potential antiviral targets. In this study, a bimolecular fluorescence complementation based screening assay is developed that can quantify a change in dimerization, using the HIV-1 accessory protein Vpr as a "proof of concept".Results demonstrated that bimolecular fluorescence complementation of Vpr could be competed off in a dose-dependent manner using untagged, full length Vpr as a competitor molecule. The change in signal intensity was measured quantitatively through flow cytometry and fluorescence microscopy in a high content screening assay. High content imaging was used to screen a library of peptides and a library of small molecules for an effect on Vpr dimerization. None of the Vpr peptides were shown to have an effect; however, one of the small molecules was shown to interfere with Vpr dimerization in a dose-dependent manner.Statement of Public Health relevance: Dimerization is a unique property of many HIV-1 viral proteins and is necessary to complete the viral life cycle, thus it has been identified as a potential drug target. By developing an assay that screens for inhibition of HIV-1 protein dimerization, high throughput screening can be performed to detect inhibitors of a new target in HIV-1 replication. Small molecules identified using this screening method could be developed into a novel anti-retroviral drug.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Zych, Courtneycourtneyzych@gmail.com
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee MemberWang, Tianyitywang@pitt.eduTYWANG
Committee MemberAyyavoo, Velpandivelpandi@pitt.eduVELPANDI
Committee MemberDi, Yuan Pupeterdi@pitt.eduPETERDI
Date: 29 June 2011
Date Type: Completion
Defense Date: 13 April 2011
Approval Date: 29 June 2011
Submission Date: 4 April 2011
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Infectious Diseases and Microbiology
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: drug screening; high content screening; HIV; Vpr; assay development; dimerization
Other ID: http://etd.library.pitt.edu/ETD/available/etd-04042011-225511/, etd-04042011-225511
Date Deposited: 10 Nov 2011 19:34
Last Modified: 15 Nov 2016 13:38
URI: http://d-scholarship.pitt.edu/id/eprint/6753

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